Method for treating convulsions and epilepsy with organic copper compounds

ABSTRACT

Copper-dependent enzymes are required for normal brain development and function. Copper deficiency can result in pathological disorders accompanied by convulsive seizures or tremors in man and animals. The present invention is directed to a method for treating convulsions or epilepsy comprising administration of a therapeutically effective amount of an organic compound of copper having anticonvulsant activity. Those compounds include copper complexes of carboxylic acids, acylsalicylates, salicylates, amino acids, imines and known anticonvulsant and antiepileptic drugs.

This application is a division of application Ser. No. 640,731 filedAug. 14, 1984, now issued as U.S. Pat. No. 4,670,428, which in turn is acontinuation-in-part of application Ser. No. 344,309 filed Feb. 1, 1982,now abandoned, which in turn is a continuation of application Ser. No.154,132 filed May 29, 1980, now abandoned.

FIELD OF THE INVENTION

The present invention relates to a method of treating convulsions,including convulsive tremors and convulsive seizures, and, inparticular, epilepsy, with organic copper compounds.

BACKGROUND OF THE INVENTION

Copper is a normal component of the human brain, which contains about370 mg of copper per gram of tissue ash. This amount of tissue copperranks second only to that amount found in the liver, the storage organfor copper.

Normal brain development and function requires a number ofcopper-dependent enzymes. The following is a list of these enzymes andtheir role in brain function.

    ______________________________________                                        Copper-Dependent Enzyme                                                                          Role in Brain Function                                     ______________________________________                                        (1) Cyctochrome c oxidase                                                                            Cellular respiration                                   (2) Cerebrocuprein (cerebral                                                                         Dismutation of superoxide                                  superoxide dismutase)                                                                            anion radicals                                         (3) Tyrosinase         Conversion of tyrosine to                                                     DOPA                                                   (4) Dopamine-β-hydroxylase                                                                      Conversion of dopamine to                                                     norepinephrine and                                                            epinephrine                                            (5) Lysyl oxidase      Conversion of procollagen                                                     to tropocollagen and                                                          proelastin to elastin in                                                      the vasculature                                        ______________________________________                                    

In addition, copper-dependent processes are required for the modulationof prostaglandin syntheses, lysosomal membrane permeability, theactivity of histamine, and myelinogenesis.

A variety of brain pathologic disorders accompanied by convulsiveseizures are associated with abnormal copper metabolism in humans. Serumcopper is elevated in epileptic patients, but brain copper levels aremarkedly reduced in autopsied epileptics. The elevated serum copperconcentrations may indicate physiologic mobilation of copper from theliver to the brain in life but depleted stores leading to copperdeficiency may account for decreased brain levels postmortem. Childrenwith severe copper deficiency due to inadequate intake or Menkes'Syndrome, which includes depleted liver copper stores and markedlydecreased brain copper levels, are known to have convulsive seizures asa constant feature of their copper deficiency. In addition, neonatalcopper deficiency and the copper deficiency associated with Menkes'Syndrome are also associated with severe or terminal central nervoussystem disorders.

Seizures as well as neuronal and cerebral degeneration also occur incopper-deficient anaimals. Both quaking mice and mottled mice exhibittremors as well as neural and central nervous system degeneration assymptoms of their genetic copper deficiency. Rats which are madecopper-deficient by removing copper from their diet also exhibitconvulsive tremors and central nervous system degeneration. Theobservation of seizures and central nervous system degeneration inassociation with a reduction in brain copper levels and concomitantreduction in norepinephrine and epinephrine levels which have beenproposed to be seizure modulators, are consistent with known copperrequirements [Jobe, P. C., A. L. Picchioni and L. Chin, Role of BrainNorepinephrine in Audiogenic Seizure in the Rat, J. Pharmac. Exp. Ther.,184: 1-10 (1973), hereby incorporated by reference]. Further, complexingagents which produce tremors in these animals also reduce brain copperlevels [Hadzovic, S., R. Kosak and P. Stern, The Effect of TremorigenicSubstances on the Copper Content of the Rat Brain, J. Neurochem, 3:1027-29 (1966); Price, T. R. and P. Silberfarb, Convulsions FollowingDisulfiram Treatment, Am. J. Psychiatry, 133: 235 (1976), herebyincorporated by reference]. Finally, lambs born to ewes living on copperdeficient pastures have a poorly developed central nervous system andexhibit tremors. On recognition, this enzootic ataxia is prevented byinjecting the pregnant ewes with copper complexes [Underwood, E. J., In:Trace Elements in Human and Animal Nutrition, 4th Ed. Academic Press,New York, pp. 56-108 (1977), hereby incorporated by reference].

Existing antiepileptic drugs have been found to be ineffective intreating many individuals with epilepsy. This is in part due to seriousside effects associated with these agents which include: intolerance,sedation, gingival hyperplasia, ataxia, nystagmus, dipolpia, vertigo,psychoses, lethargy, euphoria, mydriasis, headache, hyperactivity,confusion, hallucinations, peripheral neuropathy, gastrointestinalirritation, vomiting, nausea, epigastric pain, anorexia, increasedappetite, hypoglycemia, glycosuria, osteomalacia, symptoms of systemiclupus erythematosus, dermatoses, hepatic necrosis, many blood dyscrasiasand lymphadenopathy [Woodbury, D. M. and E. Fingl, The PharmacologicalBasis of Therapeutics, 5th Ed., MacMillan Pub., New York, pp. 201-225(1975), hereby incorporated by reference]. Ataxia, anorexia [Underwood,E. J., In: Trace Elements in Human and Animal Nutrition, Id.],peripheral neuropathy, nystagmus, lethargy, and osteomalacia areassociated with copper deficiency [Danks, D. M., Copper Transport andUtilization in Menkes' Syndrome and in Mottled Mice, Inorg. Persp. Biol.Med. 1: 73-100 (1977); Sorenson, J. R. J., Therapeutic Uses of Copper,In: Copper in the Environment, Ed. by J. O. Nriagu, John Wiley and Sons,New York, pp. 83-162 (1979); Underwood, E. J., In: Trace Elements inHuman and Animal Nutrition, Id., hereby incorporated by reference].

SUMMARY OF THE INVENTION

The present invention seeks to overcome the problems and disadvantagesof the prior art. As pointed out, supra, existing antiepileptic drugsare ineffective in treating many individuals with epilepsy because oftheir toxic side-effects. If drug-induced toxicities are in part causedby the removal of copper from some copper-dependent metalloprotein ormetalloenzyme via complexation as a result of therapy, then thesetoxicities may be avoided by treatment with a copper complex of thesedrugs. Because copper complexes are known to have potent antiulceractivity and lack gastrointestinal irritant activity [Sorenson, J. R.J., Copper Chelates As Possible Active Forms of the Anti-ArthriticAgents, J. Med. Chem. 19(1): 135-147 (1976); Sorenson, J. R. J., CopperComplexes, A Unique Class of Antiarthritic Drugs, Prog. Med. Chem. 15:211-260 (1978); and Walker, W. R., R. Reeves and D. J. Kay, Role of Cu²⁺and Zn²⁺ in Physiological-Activity of Histamine in Mice, Search 6:134-135 (1975), hereby incorporated by reference], it is conceivablethat at least the gastrointestinal side-effects of the existingantiepileptic drugs may be circumvented by using copper complexes intherapy. If copper complexes of the antiepileptic drugs or other coppercomplexes have increased anticonvulsant activities and do not causegastrointestinal irritation or the other toxicities associated with thecurrently used drugs they would offer more effective and less toxictherapy of convulsions and epilepsy.

Broadly, the present invention is directed to a method for treatingconvulsions, including convulsive tremors and convulsive seizures, andepilepsy comprising administration of a therapeutically effective amountof an organic compound of copper (in the cuprous or cupric form) havinganticonvulsant and/or antiepileptic activity.

Such compounds include but are not limited to copper complexes ofimines, including the following specific types of imines which possessdistinctive configurations when complexed with copper: bisethyleneimineSchiff bases, salicylidene-amino acid Schiff bases andpyridoxylidene-amino acid Schiff bases.

Such compounds further include but are not limited to copper complexesof carboxylic acids. Included among these carboxylic acids are arylcarboxylic acids and also branched and unbranched aliphatic carboxylicacids, for example, those carboxylic acids with aliphatic chains of oneto seven carbons in length. The aryl carboxylic acids include, but arenot limited to, acylsalicylic acids and benzoic acids. When complexedwith copper the carboxylic acids are called copper carboxylates.

The organic copper compounds useful in the practice of this inventionalso include copper complexes of amino acids. Two molecules of the sameor different amino acid complex with one atom of copper to form adistinctive copper coordination compound. The twenty common amino acidsas well as other less common amino acids are potentially useful.

The organic copper compounds that can be used in the invention includecopper complexes of salicylic acid and substituted salicylic acids. Suchsalicylic acids form copper salicylates.

One of the remarkable aspects of Applicant's invention is thedemonstration that copper complexes of salicylates, acylsalicylates andamino acids exhibit anticonvulsant and/or antiepileptic activity. ToApplicant's knowledge, no one has ever reported that salicylates,acylsalicylates, or amino acids alone, i.e., not complexed with copper,have any anticonvulsant and/or antiepileptic. On the contrary, what isknown is that salicylate and acetylsalicylate (aspirin) actually causeconvulsions at high doses, making Applicant's discovery that coppercomplexes of these compounds have anticonvulsant activity all the moreremarkable. Applicant's discovery further supports the suggestion ofreduced toxicity of copper complexes of existing anticonvulsant(antiepileptic) drugs.

Also suitable for use in the practice of this invention are coppercomplexes of known anticonvulsant/antiepileptic compounds. Suchcompounds are of the following classes: hydantoins, barbiturates,desoxybarbiturates, iminostilbenes, acetylureas, succinimides,benzodiazepines, oxazolidinediones, sulfonamides and fatty acids(saturated or unsaturated) or mixtures of any of the foregoingcompounds. Remarkably, it has been found that subcutaneousadministration of copper complexes of certain of the knownanticonvulsant drugs, specifically amobarbital, is free of side effects(hypnosis or sedation) associated with the non-copper-complexed form ofthe drug. The potential for elimination of side-effects associated withknown anticonvulsant and antiepileptic drugs by using copper complexesthereof is a particularly important discovery.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 graphically depicts the anticonvulsant activity versus time forCu(II) (salicylate)₂ in preventing the Metrazol-induced seizure aftergiving 100 mg/kg subcutaneously (closed circles) and in preventing theMaximal Electroshock-induced seizure after giving 600 mg/kgsubcutaneously (open circles).

DESCRIPTION OF THE INVENTION

Copper complexes were synthesized using reported methods [Sorenson, J.R. J., Copper Chelates As Possible Active Forms of the Anti-ArthriticAgents, Id.; U.S. Pat. No. 4,221,785 of Sorenson, J. R. J., herebyincorporated by reference].

The copper complexes were submitted to the National Institute ofNeurological and Communicative Disorders and Stroke (NINCDS) which hasan Antiepileptic Drug Development (ADD) program to screen compounds foranticonvulsant activity. Test compounds were either dissolved in 0.9%saline, or suspended in either 30% polyethylene glycol 400 in 0.9%saline or 0.2% suspending agent like polyoxyethylene glycol or Tween 80in 0.9% saline prior to injection into male Carworth Farms #1 mice orSprague-Dawley rats. Thus, compounds can be administered as solutions,suspensions or ointments. Unless otherwise specified, concentrations arepercent by weight.

In Phase I identification of anticonvulsant activity, test compoundswere given intraperitoneally (IP) at 30, 100, 300 and, in some cases,600 milligrams per kilogram (mg/kg) of body weight and protectionagainst Maximal Electroshock and/or Metrazol-induced seizures wasdetermined 30 minutes, 4 hours, or later. Initial studies using theintraperitoneal route of administration demonstrated that coppercomplexes were effective as anticonvulsants but stimulation ordepression (rotating rod toxicity) and lethality were occasionallyobserved at the highest doses given. Subsequently the routine protocolwas modified and certain of the test compounds (see Tables, infra)re-evaluated following subcutaneous (SC) injection at periods of up to 8hours post injection. Subcutaneous administration can be helpful indetermining whether or not hypnotic activity can be distinguished fromanticonvulsant activity based upon the decreased rate of absorptionassociated with this route of administration as opposed to the morerapid rate of absorption associated with IP administration. With thisprotocol none of the copper complexes so tested was found to be toxic inthe rotating rod test and no deaths were observed, even at the highestdoses given. In addition, some of these compounds were found to haveanticonvulsant activity at doses less than 30 mg/kg and for prolongedperiods of up to 6 to 8 hours post injection.

Compounds found to be effective only at 30 minutes are viewed as rapidin onset (due to rapid distribution to the central nervous system) andof short duration. Compounds that are effective only at 4 hours areviewed as slower in onset. Those compounds that are effective at both 30minutes and 4 hours are viewed to be rapid in onset and of prolongedduration. Variability in onset and duration may be useful in the designof therapeutic regimens in which combinations of compounds areadministered to a convulsant or epileptic patient.

Minimal anticonvulsant activity and the lack of toxicity were criteriarequired for Phase II studies, in which the time of peak anticonvulsanteffect in the Maximal Electroshock and Metrazol-induced seizures,efficacy (ED₅₀ values for protection against the Maximal Electroshockand Metrazol-induced seizures), and lethality (LD₅₀ in 24 hours) werequantified. All statistics were obtained by probit analyses.

SEIZURE MODELS

Maximal Electroshock Seizure Test. Maximal electroshock seizures wereelicited with a 60 cycle alternating current of 50 mA intensity (5-7times that necessary to elicit minimal electroshock seizures) deliveredfor 0.2 seconds via corneal electrodes. A drop of 0.9% saline isinstilled in the eye prior to application of the electrodes in order toprevent the death of the animal. Abolition of the hind limb tonicextension component of the seizure is defined as protection.

Subcutaneous Pentylenetetrazol (Metrazol) Seizure Threshold Test.Eighty-five mg/kg of pentylenetetrazol (produces seizures in greaterthan 97% of mice) was administered as a 0.5% solution subcutaneous inthe posterior midline. The animal was observed for 30 minutes. Failureto observe even a threshold seizure (a single episode of clinic spasmsof at least 5 seconds duration) is defined as protection.

Toxicity. The rotating rod was used to evaluate neurotoxicity. Theanimal was placed on a 1-inch diameter knurled plastic rod rotating at 6revolutions per minute. Normal mice can remain on a rod rotating at thisspeed indefinitely. Neurologic toxicity is defined as the failure of theanimal to remain on the rod for 1 minute and is due to eitherstimulation or depression of the central nervous system.

EXPERIMENTAL EXAMPLES Copper Complexes of Acylsalicylates

The data obtained for a number of copper complexes of acylsalicylatesfollowing subcutaneous (SC) administration are presented in Table I.Cu(II) refers to the cupric form of the compound, that is, copper with avalency of +2. Structures I and II schematically depict the genericstructure of an acylsalicylate copper complex (where R representshydrogen, branched or unbranched lower alkyl, aryl, alkyl-aryl, orsubstituted alkyl or aryl groups; R" represents branched or unbranchedlower alkyl, aryl, halogen, or branched or unbranched lower alkyl oraryl groups substituted with halogens or oxygen-containing substituentssuch as hydroxy or alkoxy or nitrogen-containing substituents such asamino or nitro; and L represents solvating or other ligands capable ofbonding to copper as indicated, such as water, alcohols, amines, ethers,sulfoxides and other solvents and competing ligands). The nonsolvatedform of copper complexes of acylsalicylates, i.e., in the absence of L,exhibits a central binuclear configuration (as in Structure I)characteristic of nonsolvated forms of copper complexes of carboxylicacids which associate with copper atoms via their carboxyl groups in a4:2 (carboxylic acid:copper atom) ratio. When L is present, the coppercomplex can exist in either the binuclear configuration of Structure Ior the mononuclear configuration of Structure II depending on theaffinity of L for copper. ##STR1##

                  TABLE I                                                         ______________________________________                                        ANTICONVULSANT ACTIVITY (PHASE I)                                             OF SOME COPPER COMPLEXES OF ACYLSALICYLATES                                                                Maximal                                                       %               Electro-                                         COMPLEX      Copper  Route   shock  Metrazol                                  ______________________________________                                        Cu(II).sub.2 (adamantyl-                                                                   9       .sup. SC.sup.1                                                                        .sup. I.sup.2                                                                        A.sup.3 at 30                             salicylate).sub.4                   and 300                                                                       mg/kg at                                                                      30 min.                                   Cu(II).sub.2 (acetyl-                                                                      15      SC      I      A at 100                                  salicylate).sub.4                   and 300                                                                       mg/kg at                                                                      30 min.                                                                       and 6                                                                         hrs.; A                                                                       at 30,                                                                        100 and                                                                       300 mg/kg                                                                     at 8 hrs.                                 Cu(II)       11      SC      I      A at 300                                  (acetylsalicylate).sub.2            mg/kg at                                  (dimethylsulfoxide).sub.2           30 min.;                                                                      A at 100                                                                      mg/kg at                                                                      4 hrs.                                    Cu(II)       11      SC      I      A at 300                                  (acetylsalicylate).sub.2            mg/kg at                                  (pyridine).sub.2                    30 min.;                                                                      A at 100                                                                      and 300                                                                       mg/kg at                                                                      4 hrs.                                    Cu(II).sub.2 (3,5-dibromo-                                                                 8.6     IP      A at 100                                                                             A at 100                                  acetylsalicylate).sub.4      mg/kg at                                                                             mg/kg at                                                               4 hrs. 30 min.                                                        SC      A at 300                                                                             I                                                                      mg/kg at                                                                      4 hrs.                                           Cu(II).sub.2 (3,5-diiodo-                                                                  6.8     IP      I      A at 100                                  acetylsalicylate).sub.4             mg/kg at                                  (H.sub.2 O).sub.6                   30 min.                                                                       and 4 hrs.                                                     SC      A at 300                                                                             A at 300                                                               mg/kg at                                                                             mg/kg at                                                               4 hrs. 4 hrs.                                                         .sup. SC.sup.4                                                                        Not    A at 105                                                               tested mg/kg at                                                                      30 min.                                   ______________________________________                                         .sup.1 SC = Subcutaneous.                                                     .sup.2 Inactive at doses studied.                                             .sup.3 Activity at doses and times indicated.                                 .sup.4 Phase II data.                                                    

Cu(II)₂ (adamantylsalicylate)₄ was found to have anticonvulsant activityat 30 minutes following subcutaneous injection at 30 and 300 mg/kg.Cu(II)₂ (acetylsalicylate)₄ [alternatively called copper aspirinate orCu(II)₂ (aspirinate)₄ ] was found to have activity at 30 minutes, 6hours and 8 hours following subcutaneous injection of the entire rangeof doses studied. The dimethylsulfoxide and pyridine solvates ofCu(II)(acetylsalicylate)₂ were found to be effective at the higher dosesstudied at 30 minutes and 4 hours. None of these complexes was found tohave rotating rod toxicity at any of the doses or times studiedfollowing subcutaneous injection. Cu(II)₂ (3,5-dibromoacetylsalicylate)₄and Cu(II)₂ (3,5-diiodoacetylsalicylate)₄ were also effective inprotecting against both models of seizure.

COPPER COMPLEXES OF SALICYLATES ##STR2##

A group of copper complexes of salicylates with the generic structuresshown schematically by structures III and IV (where R" and L representthe same groups, substituents, and ligands described for Structures Iand II) were also evaluated for their anticonvulsant activity. Thenonsolvated form of copper complexes of salicylates, i.e., in theabsence of L, exhibits a mononuclear configuration as in Structure III.When L is present, the copper complex can exist in either themononuclear configuration of Structure III or the binuclearconfiguration of Structure IV depending on the affinity of L for copper.

As shown in Table II, Cu(II)(salicylate₂),Cu(II)(4-tertiarybutylsalicylate)₂ andCu(II)(3,5-ditertiarybutylsalicylate)₂ were found to be effective at thehigher doses studied and for prolonged periods of up to 4 hours.Cu(II)(salicylate)₂ (pyridine)₂ had activity representative of acompound with rapid onset. The 3,5-diisopropylsalicylate,3,5-ditertiarybutylsalicylate, 3,5-dibromosalicylate and3,5-dichlorosalicylate complexes are of special interest because theywere effective in preventing both the Maximal Electroshock andMetrazol-induced seizures in Phase I studies. All of these complexeswere also found to be free of rotating rod toxicity at all of the dosesand time periods studied following subcutaneous injection. Alsopresented in Table II are data obtained for three 4-substitutedsalicylates, Cu(II)(4-nitro-salicylate)₂, Cu(II)(4-aminosalicylate)₂ andCu(II)(4-acetylaminosalicylate)₂, which were also effective inpreventing seizures. However, administration ofCu(II)(4-nitrosalicylate)₂ at 100 mg/kg, Cu(II)(4-aminosalicylate)₂ at300 mg/kg and Cu(II)(acetylaminosalicylate)₂ at 600 mg/kg elicitedrotating rod toxicity. Moreover, administration ofCu(II)(4-nitrosalicylate)₂ at doses higher than 100 mg/kg andCu(II)(4-aminosalicylate)₂ at doses higher than 300 mg/kg caused deathin some of the test groups. These deaths may have resulted from ahypnotic overdose.

                  TABLE II                                                        ______________________________________                                        ANTICONVULSANT ACTIVITY (PHASE I)                                             OF SOME COPPER COMPLEXES OF SALICYLATES                                                                    Maximal                                                       %               Electro-                                         COMPLEX      Copper  Route   shock  Metrazol                                  ______________________________________                                        Cu(II)(salicylate).sub.2                                                                   19      .sup. SC.sup.1                                                                        .sup. I.sup.2                                                                        A.sup.3 at                                                                    30, 100                                                                       and 300                                                                       mg/kg at                                                                      30 min.;                                                                      A at 300                                                                      mg/kg at                                                                      4 hrs.                                    Cu(II)(salicylate).sub.2                                                                   --      IP      I      A at 300                                  (pyridine).sub.2                    mg/kg at                                                                      30 min.                                   Cu(II).sub.2 14      SC      I      A at 300                                  (4-tertiarybutyl-                   mg/kg at                                  salicylate).sub.2.1/2H.sub.2 O      30 min.;                                                                      A at 300                                                                      and 600                                                                       mg/kg at                                                                      4 hrs.                                    Cu(II)(3,5-diisopro-                                                                       13      SC      A at 300                                                                             A at 30,                                  pylsalicylate).sub.2         mg/kg at                                                                             100 and                                                                4 hrs; 300 mg/kg                                                              A at 100                                                                             at 30 min.;                                                            and 300                                                                              A at 100                                                               mg/kg at                                                                             and 300                                                                6 hrs.;                                                                              mg/kg at                                                               A at 300                                                                             4 hrs.;                                                                mg/kg at                                                                             A at 100                                                               8 hrs. mg/kg at 6                                                                    hrs.; A at                                                                    100 and                                                                       300 mg/kg                                                                     at 8 hrs.                                 Cu(II)(3,5-ditertiary-                                                                     --      IP      A a 100                                                                              A at 30                                   butylsalicylate).sub.2       mg/kg at                                                                             and 100                                                                4 hrs. mg/kg at                                                                      30 min.                                                        SC      A at 300                                                                             A at 300                                                               mg/kg at                                                                             mg/kg at                                                               4 hrs. 4 hrs.                                    Cu(II)       --      .sup. IP.sup.4                                                                        I      A at                                      (4-nitrosalicylate).sub.2           100.sup.3                                                                     mg/kg.sup.6                                                                   at 30 min.                                Cu(II)       --      IP      I      A at 30,                                  (4-aminosalicylate).sub.2           100 and                                                                       300.sup.5                                                                     mg/kg.sup.6                                                                   at 30                                                                         min.; A                                                                       at 100                                                                        mg/kg.sup.6                                                                   at 4 hrs.                                 Cu(II)(4-acetyl-                                                                           --      IP      A at 600.sup.5                                                                       I.sup.7                                   aminosalicylate).sub.2       mg/kg at                                                                      30 min.                                          Cu(II)(5-chloro-                                                                           --      IP      I      A at 300                                  salicylate).sub.2                   mg/kg at                                                                      30 min.                                                                       and 30                                                                        mg/kg at                                                                      4 hrs.                                                         .sup. IP.sup.8                                                                        Not    A at 30                                                                tested mg/kg at                                                                      6 hrs.                                    Cu(II)(3,5-dibromo-                                                                        --      IP      A at 300                                                                             A at 30                                   salicylate).sub.2 (H.sub.2 O).sub.3                                                                        mg/kg at                                                                             mg/kg at                                                               4 hrs. 30 min.                                                                       and 100                                                                       mg/kg at                                                                      4 hrs.                                                         SC      A at 300                                                                             A at 600                                                               mg/kg at                                                                             mg/kg at                                                               4 hrs. 4 hrs.                                    Cu(II)(3,5-dichloro-                                                                       --      IP      A at 600                                                                             A at 100                                  salicylate).sub.2 (H.sub.2 O).sub.2                                                                        mg/kg at                                                                             mg/kg at                                                               4 hrs. 30 min.                                                        SC      I      A at 600                                                                      mg/kg at                                                                      4 hrs.                                    ______________________________________                                         .sup.1 SC = Subcutaneous.                                                     .sup.2 Inactive at doses studied.                                             .sup.3 Activity at doses and times indicated.                                 .sup.4 IP = Intraperitoneal.                                                  .sup.5 Rotating Rod toxicity.                                                 .sup.6 Lethal at higher doses.                                                .sup.7 Lethal at 100 mg/kg at 4 hours.                                        .sup.8 Phase II data.                                                    

PHASE II EVALUATION OF CU(II)(SALICYLATE)₂

The Phase I test data presented in Tables I, II (supra) and III (infra)were all obtained in a standardized protocol using routine doses androutine times for the qualitative evaluation of anticonvulsant effectsof the compounds tested. The NINCDS-ADD Program Phase II follow-upevaluation of active compounds with low toxicity is a quantitation ofthe anticonvulsant activity and acute toxicity. The anticonvulsantactivity is quantitated by determining the times of peak activity andED₅₀ values in the Maximal Electroshock and Metrazol-induced seizures.The acute toxicity is quantitated by determining the 24-hour LD₅₀ value.

The first copper complex selected by the ADD Program for Phase IIevaluation was Cu(II)(salicylate)₂. The data presented in FIG. I showthat the time of peak effect for the inhibition of Metrazol-inducedseizures following subcutaneous administration of 100 mg/kg was 2 hoursand the time of peak effect for the inhibition of Maximal Electroshockinduced seizures was 7 hours following subcutaneous administration of600 mg/kg.

These data point out that the times of peak effects were different fromthe time of routine evaluation in the Phase I tests and that the doserequired to protect against the Maximal Electroshock-induced seizure waslarger than the largest dose used in the routine Phase I test. The dataplotted in FIG. 1 also show that there was a rapid onset of protectionagainst the Metrazol-induced seizure which decreases over theextrapolated period of 7 hours. The onset of protection against MaximalElectroshock-induced seizure was slower but the activity was prolongedover the 24-hour period (37% inhibition at 24 hours) and beyond. TheED₅₀ values for protection against Maximal Electroshock andMetrazol-induced seizures were 360 mg/kg and 38 mg/kg, respectively. TheLD₅₀ value for this compound was 441 mg/kg. This value was not muchdifferent from the ED₅₀ for protection against MaximalElectroshock-induced seizures but it was over 10 times the ED₅₀ for theprevention Metrazol-induced seizures.

These data show that while Cu(II)(salicylate)₂ was inactive inprotecting against the Maximal Electroshock-induced seizure in routinePhase I studies, it was found to be active in protecting against thisseizure when higher doses were used and the activity was evaluated atdifferent time periods. As a result, inactivity in Phase I studiescannot be taken as evidence of no activity at any time period or higherdoses. The possibility exists that these apparently inactive compoundsmay be active in protecting against seizures when the treatment protocolis modified to include prolonged pretreatment (i.e., longer periods oftime after administration of a copper complex but before inducement ofthe seizure) and/or higher doses.

COPPER COMPLEXES OF AMINO ACIDS

A series of copper complexes of bidentate amino acids genericallydepicted by Structure V (where R represents the alpha substituents ofthe D or L amino acids, wherein D and L represent the configuration ofthe alpha carbon and where two of the same or two different amino acidsform the complex and L represents solvating or other ligands such aswater, alcohols, amines, ethers, sulfoxides and other solvents andcompeting ligands) and a copper complex of a tridentate amino acid(glutamic acid) were evaluated as anticonvulsants. The results arepresented in Table III. ##STR3##

                  TABLE III                                                       ______________________________________                                        ANTICONVULSANT ACTIVITY (PHASE I)                                             OF SOME COPPER COMPLEXES OF AMINO ACIDS                                                                     Maximal                                                       %               Electro-                                        COMPLEX       Copper  Route   shock  Metrazol                                 ______________________________________                                        Cu(II)        22      .sup. IP.sup.1                                                                        .sup. NT.sup.2                                                                       A.sup.3 at                               (L-threoninate)                      30, 100 and                              (L-serinate)                         300 mg/kg                                                                     at 30                                                                         min. and                                                                      4 hrs.                                   Cu(II)        24      IP      NT     A at 30,                                 (L-threoninate)                      100 and                                  (L-alaninate)                        300 mg/kg                                                                     at 30                                                                         min. and                                                                      4 hrs.                                   Cu(II)        20      IP      NT     A at 30,                                 (L-valinate).sub.2 H.sub.2 O         100 and                                                                       300 mg/kg                                                                     at 30                                                                         min. and                                                                      4 hrs.                                   Cu(II)        20      IP      NT     A at 30,                                 (L-threoninate).sub.2 H.sub.2 O      100 and                                                                       300 mg/kg                                                                     at 30                                                                         min. and                                                                      4 hrs.                                   Cu(II)        27      IP      NT     A at 30,                                 (L-alaninate).sub.2                  100 and                                                                       300 mg/kg                                                                     at 30                                                                         min. and                                                                      4 hrs.                                   Cu(II)        16      IP      NT     At at 30,                                (L-phenylalaninate).sub.2            100 and                                                                       300 mg/kg                                                                     at 30 min.                               Cu(II)        20      IP      NT     At at 100                                (L-cystinate).sub.2 H.sub.2 O        and 300                                                                       mg/kg at                                                                      30 min.                                  Cu(II)        --      .sup. SC.sup.1                                                                        NT     A at 30,                                 (L-serinate).sub.2                   100 and                                                                       300 mg/kg                                                                     at 30                                                                         min. and                                                                      4 hrs..sup.6                             Cu(II)        --      IP      .sup. I.sup.5                                                                        A at 300                                 (L-tryptophanate).sub.2              and 600                                                                       mg kg at                                                                      30 min.                                                                       and 4 hrs.                               Cu(II)        --      SC      I      A at 600                                 (L-glutamate).sub.2                  mg/kg at                                                                      4 hrs.                                                         IP      I      A at                                                                          100.sup.7                                                                     mg/kg at                                                                      30 min; A                                                                     at 30 and                                                                     100 mg/kg                                                                     at 4 hrs.                                Cu(II)        --      SC      I      I                                        (L-leucinate).sub.2   IP      I      A at 100                                                                      mg/kg at                                                                      30 min.                                                                       and A at                                                                      30 and                                                                        100 mg/kg                                                                     at 4 hrs.                                Cu(II)        --      SC      I      A at 300                                 (L-isoleucinate).sub.2               and 600                                                                       mg/kg at                                                                      30 min.                                                                       and 600                                                                       mg/kg at                                                                      4 hrs.                                   Cu(II)(L-isoleucinate).sub.2                                                                        IP      A at 600.sup.6                                                                       A at                                                                   mg/kg at                                                                             100.sup.6                                                              30 min.                                                                              mg/kg                                                                         at 30                                                                         min.                                     ______________________________________                                         .sup.1 Not Tested.                                                            .sup.2 Active at doses and times indicated.                                   .sup.3 Rotating red toxicity observed with 300 mg/kg at 4 hrs.                .sup.4 Inactive at 30, 100, 300 and 600 milligrams per kilogram at 30 min     and 4 hrs.                                                                    .sup.5 Rotating rod toxicity.                                                 .sup.6 Lethal at higher doses.                                           

Cu(II)(L-threoninate)(L-serinate), Cu(II)(L-threoninate)(L-alaninate),Cu(II)(L-valinate)₂, Cu(II)(L-threoninate)₂ and Cu(II)(L-alaninate)₂were found to be effective against the Metrazol-induced seizure at alldoses studied and at both time intervals, 30 minutes and 4 hours.Rotating rod toxicity was observed with the first four of thesecompounds but only at the highest dose studied (300 mg/kg) at the end ofthe 4-hour observation. Cu(II)(L-phenylalaninate)₂ andCu(II)(L-cystinate)₂ were also effective against the Metrazol-inducedseizure at all three doses studied but only at the shorter time period.

Cu(II)(L-serinate)₂, Cu(II)(L-tryptophanate)₂, Cu(II)(L-glutamate)₂,Cu(II)(L-leucinate)₂ and Cu(II)(L-isoleucinate)₂ have also been found tohave anticonvulsant activity. In all cases these compounds were found tobe more effective following IP than SC administration but they were alsomore toxic following IP administration. This is consistent with a morerapid absorption of greater amounts of these compounds following IPadministration. The lethality associated with higher doses of some ofthe compounds is consistent with the possibility that these animals hadbeen given hypnotic overdoses. The isoleucine complex was unique becauseit inhibited both the Maximal Electroshock and Metrazol-induced seizuresin these Phase I tests.

COPPER COMPLEXES OF IMINES

Copper complexes of imines were evaluated for their anticonvulsantactivity. The types of imine compounds tested included copper complexesof the following: (a) salicylidene-amino acid Schiff bases, the genericstructure of which is depicted by Structure VI (where R represents thealpha substituents of the D or L amino acids, D and L denoting theconfiguration of the alpha carbon; R" represents branched or unbranchedlower alkyl, aryl or halogen groups, or branched or unbranched loweralkyl or aryl groups substituted with halogens, oxygen-containinggroups, e.g., hydroxy or alkoxy, or nitrogen-containing groups, e.g.,amino or nitro; and L represents solvating or other ligands capable ofbonding to copper as indicated, such as water, alcohols, amines, ethers,sulfoxides, and other solvents and competing ligands); (b)bisethyleneimine Schiff bases, the generic structures of which aredepicted by Structures VII and VIII (where R and R" represent branchedor unbranched lower alkyl, aryl or halogen groups, or branched orunbranched lower alkyl or aryl groups substituted with halogens,oxygen-containing groups, e.g., hydroxy or alkoxy, ornitrogen-containing groups, e.g., amino or nitro; and L representssolvating or other ligands capable of bonding with copper as indicatedsuch as water, alcohols, amines, ethers, sulfoxides, and other solventsand competing ligands); and (c) pyridoxylidene-amino acid Schiff bases,the generic structure of which is depicted by Structure IX (where Rrepresents the alpha substituents of the D or L amino acids; D and Ldenoting the configuration of the alpha carbon; and L representssolvating or other ligands capable of bonding with copper as indicatedsuch as water, alcohols, amines, ethers, sulfoxides, and other solventsand competing ligands). ##STR4##

                  TABLE IV                                                        ______________________________________                                        ANTICONVULSANT ACTIVITY (PHASE I)                                             OF SOME COPPER COMPLEXES OF SALICYLIDENE-                                     AMINO ACID AND BISETHYLENEIMINE SCHIFF BASES                                                       Maximal                                                                       Electro-                                                 COMPLEX      Route   shock    Metrazol                                        ______________________________________                                        Cu(II)       SC      I        A (slight) at 100                               Salicylidene-L-               and 300 mg/kg.sup.2                             valinate                      at 4 hrs.                                                    IP      I        A at 100 mg/kg.sup.2                                                          at 30 min. and                                                                30 mg/kg.sup.2 at                                                             4 hrs.                                          Cu(II)       SC      I        A (slight) at 600                               Salicylidene-L-               mg/kg at 30 min.                                histidinate  IP      I        I                                               Cu(II)       SC      A at 100 A at 100, 300'                                  Bisacetylacetonethyl-                                                                              mg/kg at and 600' mg/kg                                  eneimine             30 min.; at 4 hrs.                                                            A at 100,                                                                     300' and                                                                      600' at                                                                       4 hrs.                                                   Cu(II)       SC      I        I                                               bissalicylato-                                                                             IP      I        A (marginal) at                                 ethyleneimine                 300 and 600 mg/kg                                                             at 4 hrs.                                       ______________________________________                                         A = Active.                                                                   I = Inactive at doses studied.                                                IP = Intraperitoneal.                                                         SC = Subcutaneous.                                                            .sup.1 Rotating rod toxicity.                                                 .sup.2 Lethal at higher doses.                                           

The data obtained with copper complexes of salicylidene-amino acidSchiff bases and bisethyleneimine Schiff bases are presented in TableIV. The majority of these copper complexes had weak activity. Thesalicylidene-L-valinate complex also caused lethality at higher doses.The bisacetylacetonethyleneimine complex was effective in preventingboth Maximal Electroshock and Metrazol-induced seizures, but rotatingrod toxicity, which may also have been due to hypnotic activity, wasfound at the higher doses studied.

The data obtained with copper complexes of pyridoxylidene-amino acidSchiff bases are presented in Table V. These data show that thepyridoxylideneglycinate complex protected against both types of seizure.The serinate, tryptophanate and threoninate complexes were moreeffective but only protected against the Metrazol-induced seizure. Thephenylalaninate and valinate complexes had no activity.

Some of these complexes are as active or more active than existingantiepileptic drugs. Other potentially useful copper complexes ofpyridoxylideneamino acid Schiff bases include complexes of thefollowing: tyrosine, dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan,glutamic acid, gamma aminobutyric acid, aspartic acid, and beta-alanine.

                  TABLE V                                                         ______________________________________                                        PHASE I ANTICONVULSANT DATA OF                                                PYRIDOXYLIDENEAMINOACID COPPER COMPLEXES                                                       Challenge                                                                             Seizure Model.sup.1                                  Complex       Route    Time      MES  Metrazol                                ______________________________________                                        Cu(II)Pyridoxylidene-                                                                       IP       30     min  300  100                                   glycinate(H.sub.2 O).sub.1.5                                                                         4      hrs  I    I                                     Cu(II)Pyridoxylidene-L-                                                                     IP       30     min  I    100                                   serinate(H.sub.2 O)    4      hrs  I    100                                   Cu(II)Pyridoxylidene-L-                                                                     IP       30     min  I    30                                    tryptophanate(H.sub.2 O).sub.2                                                                       4      hrs  I    30                                    Cu(II)Pyridoxylidene-L-                                                                     IP       30     min  I    30                                    threoninate(H.sub.2 O).sub.2                                                                         4      hrs  I    30                                    Cu(II)Pyridoxylidene-L-                                                                     IP       30     min  I    I                                     phenylalaninate(H.sub.2 O).sub.2                                                                     4      hrs  I    I                                     Cu(II)Pyridoxylidene-L-                                                                     IP       30     min  I    I                                     valinate(H.sub.2 O)    4      hrs  I    I                                     ______________________________________                                         .sup.1 The numerical values are the lowest active doses in mg/kg of body      weight; I = Inactive; MES = Maximal Electroshock.                        

COPPER COMPLEXES OF CARBOXYLIC ACIDS

Several copper complexes of carboxylic acids have been tested for theiranticonvulsant activity. The carboxylic acids include branched andstraight chain aliphatic carboxylic acids as well as aryl carboxylicacids. In their nonsolvated state (i.e, in the absence of L), suchcarboxylic acids form a characteristic binuclear complex of copper asschematically depicted by Structure X (where R represents alkyl, aryl,aryl-alkyl groups wherein substituents may be hydrogen, halogen,oxygen-containing, e.g., hydroxy or alkoxy, or nitrogen-containing,e.g., amino or nitro substituents; and L represents solvating or otherligands capable of bonding to copper as indicated such as water,alcohols, amines, ethers, sulfoxides, and other solvents and competingligands). When L is present, the copper complex of the carboxylic acidcan exist in either the binuclear configuration of Structure X or themononuclear configuration of Structure X-A depending on the affinity ofL for copper. It should be recalled from the section on copper complexesof acylsalicylates, supra, that these compounds are also carboxylicacids that complex with copper in the nonsolvated state to form abinuclear coordination compound and, when in the solvated state, complexwith copper in either the binuclear or mononuclear configurations shownby Structures I and II, respectively. Similarly, fatty acids, whichinclude saturated and unsaturated monocarboxylic acids of up to aboutC₁₉ in length, can complex in either the binuclear or mononuclearconfigurations in their solvated states. ##STR5##

Copper acetate, Cu(II)₂ (acetate)₄, a binuclear copper complex of a C₂,straight chain, aliphatic carboxylic acid, was evaluated foranticonvulsant activity in a test in which aspirin (not complexed withcopper) was also evaluated. It is known that in high doses aspirin (andother salicylates) have toxic effects on the central nervous system,including convulsions [Goodman and Gilman (Eds.), The PharmacologicalBasis of Therapeutics (1980), 6th ed., MacMillan, New York, p. 689].

As shown in Table VI, aspirin was found to be inactive at 30, 100, 300and 600 mg/kg in both seizure models at both time intervals, 30 minutesand 4 hours. Because it was thought that a copper-containing compoundinjected subcutaneously at the same site as the site of subcutaneousMetrazol injection might somehow, through a direct interaction withMetrazol perhaps, prevent the induction of seizures with Metrazol, andas a result cause an apparent anticonvulsant effect, this aspect ofadministration was investigated.

                  TABLE VI                                                        ______________________________________                                        ANTICONVULSANT ACTIVITY OF                                                    ASPIRIN AND COPPER ACETATE                                                                     Seizure Model                                                                       Maximal                                                Compound      Route    Electroshock                                                                             Metrazol                                    ______________________________________                                        Aspirin       IP       .sup. I.sup.1                                                                            I.sup.1                                     Cu(II).sub.2 (acetate).sub.4                                                                 SC*     .sup. I.sup.1                                                                            A at 30,                                                                      100, 300                                                                      and 600                                                                       mg/kg at                                                                      30 min.                                                                       and 4 hrs.                                  Cu(II)(Metrazol)Cl.sub.2                                                                    IP       I          A at 30                                                                       mg/kg at                                                                      30 min.                                                   SC       I          A at 300                                                                      mg/kg at                                                                      4 hrs.                                      Cu(II).sub.2 (acetate).sub.4                                                                SC       NT         I.sup.1                                     Cu(II).sub.2 (acetate).sub.4                                                                IP         I.sup.1,2,3                                                                            A at 30                                                                       mg/kg at                                                                      30 min..sup.4,3                             Cu(II).sub.2 (acetate).sub.4                                                                IP       NT         A at 10                                                                       and                                                                           20 mg/kg                                                                      at 30                                                                         min. and                                                                      A at 5,                                                                       10 and 20                                                                     mg/kg at                                                                      4 hrs.                                      Cu(II).sub.2 (acetate).sub.4                                                                IG       .sup.  I.sup.1                                                                           I.sup.1                                     ______________________________________                                         A = Active; NT = Not Tested.                                                  IP = Intraperitoneal.                                                         SC = Subcutaneous at site different from injection of Metrazol.               IG = Intragastric.                                                            *Same injection site used for injection of Metrazol.                          .sup.1 Inactive 30, 100, 300 and 600 mg/kg at 30 min. and 4 hrs.              .sup.2 Rotating rod toxicity at 30, 100, 300 and 600 mg/kg.                   .sup.3 Lethal at 30, 100, 300 and 600 mg/kg at 4 hrs.                         .sup.4 Lethal at 100, 300 and 600 mg/kg at 30 min.                       

As shown in Table VI, Cu(II)₂ (acetate)₄ was active when injected at thesame site as the site of Metrazol injection and the copper complex ofMetrazol, Cu(II)(Metrazol)(Cl₂), had some anticonvulsant activity wheninjected at a site different from the site of Metrazol injection. Inaddition, the Metrazol complex produced no rotating rod toxicity atdoses up to 600 mg/kg, which is remarkable since Metrazol is a potentcentral nervous system stimulant accounting for its seizure producingcapacity. Such stimulation produces marked rotating rod toxicity.

The lack of anticonvulsant activity of Cu(II)₂ (acetate)₄ when it isgiven subcutaneously at an alternate site of injection is consistentwith the lack of anticonvulsant effect following subcutaneous injectionof either copper acetate or copper chloride at a site different from thesite of Metrazol injection as reported previously [Sorenson et al.,Anticonvulsant Activity of Some Copper Complexes, In: Trace Substancesin Environmental Health-XIII, D. D. Hemphill, ed., University ofMissouri Press, Columbia, Mo., pp. 360-367 (1979); U.S. patentapplication Ser. No. 344,309 filed Feb. 1, 1982]. There it was reportedthat no anticonvulsant activity was found with either copper acetate orcopper chloride using doses of 50, 100 and 300 mg/kg at 0.75, 1.5 and 3hours post subcutaneous injection at a site different from the site ofMetrazol injection.

On the the other hand, the observation of anticonvulsant activity at avery low dose, 5 mg/kg, following intraperitoneal administration ofCu(II)₂ (acetate)₄ at a site different from the subcutaneousadministration of Metrazol, suggests that copper acetate is effective ininhibiting Metrazol-induced seizures by some mechanism other than adirect interaction with Metrazol when the rate of absorption and theamount of compound absorbed from the site of administration isincreased, as it is with intraperitoneal administration. Consistently,copper acetate was found to be inactive when administered orally, theroute which is likely to provide the slowest rate of absorption and thesmallest amount of compound absorbed, in comparison with thesubcutaneous and intraperitoneal routes of administration. It may,however, be that a method of oral dosing can be developed to produceanticonvulsant activity following oral treatment with copper complexesusing prolonged treatment or facilitating oral absorption.

Other copper complexes of carboxylic acids which have been tested foranticonvulsant activity are the following: (1) Cu(II)₂ (valproate)₄,also known as Cu(II)₂ (dipropylacetate)₄, which is the binuclear coppercomplex of valproic acid, a known anticonvulsant drug (valproic acid isa C₇, branched chain, aliphatic carboxylic acid); (2) Cu(II)₂(phenylacetate)₄, a binuclear copper complex of an aliphatic carboxylicacid; and (3) Cu(II)₂ (benzoate)₄, a binuclear copper complex of an arylor aromatic carboxylic acid. The data obtained with these compounds arepresented in Table VII. Cu(II)₂ (valproate)₄ and Cu(II)₂(phenylacetate)₄ were effective in protecting against Metrazol-inducedseizures while Cu(II)₂ (benzoate)₄ was effective in protecting againstboth Maximal Electroshock and Metrazol-induced seizures.

                  TABLE VII                                                       ______________________________________                                        ANTICONVULSANT ACTIVITY OF                                                    COPPER COMPLEXES OF CARBOXYLIC ACIDS                                                        Seizure Model                                                                       Maximal                                                   Complex    Route    Electroshock                                                                              Metrazol                                      ______________________________________                                        Cu(II).sub.2                                                                             IP       I           A at 100 mg/kg                                (valproate).sub.4               at 30 min.                                               SC       I           A at 600 mg/kg                                                                at 4 hr.                                      Cu(II).sub.2                                                                             IP       I           A at 300 mg/kg                                (phenylacetate).sub.4           at 4 hr..sup.1                                Cu(II).sub.2                                                                             IP       A at 300    A at 30, 100.sup.1                            (benzoate).sub.4    mg/kg at    and 300.sup.1 mg/kg                                               30 min..sup.1                                                                             at 30 min.                                    ______________________________________                                         .sup.1 Rotating rod toxicity at 300 mg/kg.                               

COPPER COMPLEXES OF KNOWN ANTICONVULSANT AND ANTIEPILEPTIC DRUGS

Numerous compounds are known to have anticonvulsant and antiepilepticactivity. Some of the better known therapeutic agents, listed in TableVIII, fall into the following classes: hydantoins, barbiturates,desoxybarbiturates, iminostilbenes, acetylureas, succinimides,benzodiazepines, oxazolidinediones, sulfonamides and fatty acids. [SeeK. W. Leal and A. S. Troupin, Clinical Pharmacology of Anti-epilepticDrugs: A Summary of Current Information, Clin. Chem. 23: 1964-1968(1977), hereby incorporated by reference.] Copper complexes of theforegoing anticonvulsant and antiepileptic drugs can be used in thepractice of the present invention.

                  TABLE VIII                                                      ______________________________________                                        KNOWN ANTICONVULSANT                                                          AND ANTIEPILEPTIC DRUGS                                                       Class           Example                                                       ______________________________________                                        Hydantoins      Phenytoin (Dilantin)                                                          Desmethylmephenytoin                                                          Desethylethotoin                                                              5-Ethyl-5-phenylhydantoin                                     Barbiturates    Phenobarbital                                                                 Mephobarbital                                                                 Metharbital                                                   Thiobarbiturates                                                                              Thiopental                                                    Desoxybarbiturates                                                                            Primidone                                                     Iminostilbenes  Carbamazepine                                                 Acetylureas     Phenacemide                                                   Succinimides    Desmethylmethsuximide                                                         Ethosuximide                                                                  Desmethylphensuximide                                                         α-Methyl-α-phenylsuccinimide                      Benzodiazepines Chlorazepam                                                                   Desmethyldiazepam                                                             Diazepam                                                                      Chlorazepate                                                                  Chlordiazepoxide                                                              Oxazepam                                                      Oxazolidinediones                                                                             Desmethyltrimethadione                                                        Desmethylparamethadione                                       Sulfonamides    Acetazolamide                                                 Fatty Acids     Sodium Valproate                                              ______________________________________                                    

The general structure of hydantoins, barbiturates and thiobarbituratesis depicted schematically by Structure XI. ##STR6##

For hydantoins, X represents --NH--, R and R' are branched or unbranchedlower alkyl groups, aryl groups or branched or unbranched lower alkyl oraryl groups, which may be substituted with halogen or oxygen-containing(e.g., hydroxy or alkoxy) and nitrogen-containing (e.g., amino or nitro)substituents. L represents solvating or other ligands capable of bondingwith copper as indicated such as water, alcohols, amines, ethers,sulfoxides and other solvents and competing ligands.

For barbiturates, X represents ##STR7## and R, R' and L are as describedfor hydantoins.

For thiobarbiturates, sulfur replaces oxygen in bonding to copper asillustrated for barbiturates.

For oxazolidinediones, X represents --O-- and R, R' and L are asdescribed for hydantoins.

For succinimides, X represents --CH₂ -- and R, R' and L are as describedfor hydantoins.

For acetylureas, X represents NH₂ and is not bonded to carbon-5 to givean acyclic ligand. R, R' and L are as described for hydantoins.

For desoxybarbiturates the general structure is ##STR8## and R, R' and Lare as described for hydantoins.

For iminostilbenes the general structures may be ##STR9## where R and R'are the same or as described for hydantoins and L is as described forhydantoins.

For benzodiazepines the general structure may be ##STR10## where R andR' are hydrogen, halogen or nitro substituents, R" is hydrogen, alkyl,or the amide group ##STR11## is replaced by a group yielding an amidegroup on hydrolysis in vivo, and L is as described for hydantoins. Thebonding atom is Structure XV may be replaced with N-oxide, ##STR12## InStructure XVI, X may be an oxygen of an N-oxide. The C-3 carbon may alsobe substituted with a carboxyl or hydroxyl group.

For Sulfonamides, the general structure is ##STR13## where R representsaryl, alkyl-aryl, a heterocycle or substituted aryl, alkyl-aryl orheterocycle wherein the substituents are halogen or oxygen-containing(e.g., hydroxy or alkoxy) or nitrogen-containing (e.g., amino or nitro)substituents. R' may be H or the same as R. L is as described forhydantoins.

With the hypothesis that copper complexes of the antiepileptic drugsmight be the active metabolites of these drugs, several copper complexesof known antiepileptic drugs were synthesized and tested for theiranticonvulsant activity. The first series of tests were performed withthe copper complex of amobarbital and the results indicated that thecopper complex of this known anticonvulsant drug was a more potentanticonvulsant than sodium (Na) amobarbital. The data are presented inTable IX.

                  TABLE IX                                                        ______________________________________                                        COMPARISON OF THE SODIUM AND COPPER                                           AMOBARBITAL ANTICONVULSANT IN THE MAXIMAL                                     ELECTROSHOCK SEIZURE MODEL FOLLOWING                                          INTRAPERITONEAL INJECTION                                                                  Number            Average                                                                              Pro-                                                 of                Sleep  tection.sup.1                                        Animals  Dose in  Time,  Against                                 Compound     Treated  mg/kg    minutes                                                                              Seizure                                 ______________________________________                                        Na amobarbital                                                                             5        65       0       0                                      Cu(II)(amobarbital).sub.2                                                                  5        65       16.sup.2                                                                             100                                     ______________________________________                                         .sup.1 Percent protected.                                                     .sup.2 All animals slept.                                                

Subsequently, the anticonvulsant activity of copper complexes ofdilantin, valproate, phenobarbital (and pyridine and imidazole solvatesthereof), amobarbital, lorazepam, α-methyl-α-phenylsuccinimide,carbamazepine, clonazepam, oxazepam, 5-ethyl-5-phenylhydantoin,thiopental and diazepam was investigated in Phase I testing, and forthose compounds found to be active, Phase II testing. The results arepresented in Table X.

                  TABLE X                                                         ______________________________________                                        PHASE I AND SOME PHASE II ANTICONVULSANT DATA                                 FOR COPPER COMPLEXES OF ANTIEPILEPTIC DRUGS                                                          Seizure Model.sup.1                                                           Challenge       Metra-                                 Compound       Route.sup.2                                                                           Time      MES   zol                                    ______________________________________                                        Cu(II)(Dilantin).sub.2                                                                       IP      30     min  30    100                                  (H.sub.2 O).sub.3      4      hrs  30    100                                  Cu(II)(Dilantin).sub.2                                                                       .sup. IP.sup.3                                                                        4      hrs.sup.4                                                                          13    NT.sup.5                             (H.sub.2 O).sub.3                                                             Dilantin       .sup. IP.sup.3                                                                        1      hr.sup.4                                                                           7     Poten-                                                                        tiates                                                                        Metra-                                                                        zol                                                                           sei-                                                                          zures                                Cu(II).sub.2 (Valproate).sub.4                                                               IP      30     min  I     100                                                         4      hrs  I     I                                    Cu(II).sub.2 (Valproate).sub.4                                                               SC      30     min  I     I                                                           4      hrs  I     600                                  Valproic Acid  .sup. IP.sup.3                                                                        15     min.sup.4                                                                          272   149                                  Cu(II)(Phenobarbital).sub.2                                                                  IP      30     min  30    5                                    (H.sub.2 O).sub.5.5    4      hrs  30    5                                    Cu(II)(Phenobarbital).sub.2                                                                  SC      30     min  30    30                                   (H.sub.2 O).sub.5.5    4      hrs  30    30                                   Cu(II)(Phenobarbital).sub.2                                                                  IP      30     min  30    5                                    (H.sub.2 O).sub.3      4      hrs  30    5                                    Cu(II)(Phenobarbital).sub.2                                                                  SC      30     min  30    30                                   (H.sub.2 O).sub.3      4      hrs  30    30                                   Cu(II)(Phenobarbital).sub.2                                                                  .sup. IP.sup.3                                                                        2      hrs.sup.4                                                                          16    10                                   (H.sub.2 O).sub.3                                                             Cu(II).sub.n (Phenobarbital).sub.n                                                           SC      30     min  100   30                                   (H.sub.2 O).sub.2n (H.sub.2 O).sub.3n                                                                4      hrs  30    30                                   Phenobarbital  .sup. IP.sup.3                                                                        1      hr.sup.4                                                                           22    13                                   Cu(II)(Phenobarbital).sub.2                                                                  IP      30     min  100   30                                   (pyridine).sub.2       4      hrs  100   30                                   Cu(II)(Phenobarbital).sub.2                                                                  SC      30     min  I     I                                    (pyridine).sub.2       4      hrs  30    30                                   Cu(II)(Phenobarbital).sub.2                                                                  .sup. IP.sup.3                                                                        2      hrs.sup.4                                                                          17    9                                    (pyridine).sub.2                                                              Cu(II).sub.n (Phenobarbital).sub.n                                                           SC      30     min  100   30                                   (pyridine).sub.2n (H.sub.2 O).sub.3n                                                                 4      hrs  30    30                                   Cu(II).sub.n (Phenobarbital).sub.n                                                           SC      30     min  30    30                                   (pyridine).sub.2n (H.sub.2 O).sub.3n                                                                 4      hrs  100   30                                   Cu(II)(Phenobarbital).sub.2                                                                  IP      30     min  100   300                                  (imidazole).sub.2      4      hrs  100   30                                   Cu(II)(Phenobarbital).sub.2                                                                  SC      30     min  I     100                                  (imidazole).sub.2      4      hrs  100   100                                  Na.sub.2 [Cu(II)(Pheno-                                                                      IP      30     min  30    30                                   barbital).sub.4 ](H.sub.2 O).sub.2                                                                   4      hrs  100   100                                  Na.sub.2 [Cu(II)(Pheno-                                                                      SC      30     min  30    30                                   barbital).sub.4 ](H.sub.2 O).sub.2                                                                   4      hrs  30    100                                  Na.sub.2 [Cu(II)(Pheno-                                                                      .sup. IP.sup.3                                                                        6      hrs.sup.4                                                                          25    20                                   barbital).sub.4 ](H.sub.2 O).sub.2                                            Cu(II)(Amobarbital).sub.2                                                                    IP      30     min  300   30                                   (H.sub.2 O).sub.2.5    4      hrs  I     I                                    Cu(II)(Amobarbital).sub.2                                                                    IP      30     min  100   100                                  (H.sub.2 O).sub.2      4      hrs  I     I                                    Cu(II)(Amobarbital).sub.2                                                                    SC      30     min  300   100                                  (H.sub.2 O).sub.2      4      hrs  I     I                                    Cu(II)(Amobarbital).sub.2                                                                    .sup. IP.sup.3                                                                        30     min.sup.4                                                                          87    87                                   (H.sub.2 O).sub.2.5                                                           Amobarbital    IP      30     min  30    100                                                         4      hrs  I     I                                    Amobarbital    .sup. IP.sup.3                                                                        15     min.sup.4                                                                          45    53                                   Cu(II)(Amobarbital).sub.2                                                                    IP      30     min  300   100                                  (pyridine).sub.2       4      hrs  300   30                                   Cu(II)(Amobarbital).sub.2                                                                    SC      30     min  I     600                                  (pyridine).sub.2       4      hrs  100   600                                  Cu(II)(Amobarbital).sub.2                                                                    IP      30     min  600   300                                  (imidazole).sub.2      4      hrs  I     I                                    Cu(II)(Amobarbital).sub.2                                                                    SC      30     min  I     I                                    (imidazole).sub.2      4      hrs  I     600                                  Cu(II)(Lorazepam).sub.2                                                                      IP      30     min  20    I                                    (Cl).sub.2 H.sub.2 O   4      hrs  30    I                                    Cu(II)(Lorazepam).sub.2                                                                      SC      30     min  20    I                                    (Cl).sub.2 H.sub.2 O   4      hrs  100   I                                    Lorazepam      .sup. IP.sup.3                                                                        1      hr.sup.4                                                                           24    0.02                                 Cu(II)(Diazepam).sub.2 (Cl).sub.2                                                            IP      30     min  30    30                                                          4      hrs  30    30                                   Diazepam       .sup. IP.sup.3                                                                        1      hr.sup.4                                                                           19    0.17                                 Cu(II)α-methyl-α-phenyl-                                                         IP      30     min  100   100                                  succinimide(H.sub.2 O).sub.0.75                                                                      4      hrs  I     100                                  Cu(II)α-methyl-α-phenyl-                                                         SC      30     min  30    30                                   succinimide(H.sub.2 O).sub.0.75                                                                      4      hrs  100   600                                  Cu(II)Carbamaze-                                                                             IP      30     min  10    30                                   pine(H.sub.2 O).sub.2  4      hrs  100   I                                    Cu(II)Carbamaze-                                                                             SC      30     min  30    300                                  pine(H.sub.2 O).sub.2  4      hrs  100   300                                  Carbamazepine  .sup. IP.sup.3                                                                        --          9     Poten-                                                                        tiates                                                                        Metra-                                                                        zol                                                                           sei-                                                                          zures                                Cu(II)(Clonazepam).sub.2 (Cl).sub.2                                                          IP      30     min  10    1                                                           4      hrs  I     1                                    Cu(II)(Clonazepam).sub.2 (Cl).sub.2                                                          SC      30     min  1     1                                                           4      hrs  30    1                                    Cu(II)(Clonazepam).sub.2 (Cl).sub. 2                                                         .sup. IP.sup.3                                                                        30     min.sup.4                                                                          25    0.05                                                                    (30   1                                                                       min).sup.4                                 Clonazepam     .sup. IP.sup.3                                                                        --          19    0.2                                  Cu(II)oxazepam IP      30     min  10    1                                                           4      hrs  I     10                                   Cu(II)5-ethyl-5-phenyl-                                                                      IP      30     min  100   300                                  hydantoin(H.sub.2 O).sub.2.5                                                                         4      hrs  100   100                                  Cu(II)5-ethyl-5-phenyl-                                                                      SC      30     min  100   30                                   hydantoin(H.sub.2 O).sub.2.5                                                                         4      hrs  30    100                                  Cu(II)5-ethyl-5-phenyl-                                                                      IP      30     min  100   100                                  hydantoin(CH.sub.3 OH) 4      hrs  100   I                                    Cu(II)5-ethyl-5-phenyl-                                                                      SC      30     min  100   100                                  hydantoin(CH.sub.3 OH) 4      hrs  30    300                                  Cu(II)5-ethyl-5-phenyl-                                                                      IP      30     min  30    30                                   hydantoin(HO)(Cl)      4      hrs  I     I                                    (CH.sub.3 OH)                                                                 Cu(II)(N--thiopental).sub.2                                                                  IP      30     min  I     I                                    (H.sub.2 O).sub.2.5    4      hrs  I     I                                    Cu(II)(N--thiopental).sub.2                                                                  SC      30     min  I     I                                    (H.sub.2 O).sub.2.5    4      hrs  I     30                                   Cu(II)(S--thiopental).sub.2                                                                  IP      30     min  I     I                                    (H.sub.2 O).sub.2      4      hrs  I     I                                    Cu(II)(S--thiopental).sub.2                                                                  SC      30     min  I     I                                    (H.sub.2 O).sub.2      4      hrs  I     I                                    ______________________________________                                         .sup.1 The numerical values are the lowest doses in milligrams per            kilogram of body weight, I = inactive, MES = maximal electroshock.            .sup.2 IP = intraperitoneal, SC = subcutaneous.                               .sup.3 Phase II data.                                                         .sup.4 Time of peak activity in Phase II studies and ED.sub.50 values for     inhibition of seizures.                                                       .sup.5 Clonic seizures inhibited but animals stimulated by Metrazol           (continuous running).                                                    

The data provided in Table X for the inhibition of Maximal electroshockand Metrazol-induced seizures are the lowest effective doses (mg/kg). Ifa compound is found to be effective and nontoxic in Phase I evaluations,which are done to detect anticonvulsant activity, it is further examinedin Phase II studies to determine time of peak effect and ED₅₀. SincePhase II evaluations are done only after Phase I, there are Phase IIdata for a smaller number of compounds. There are no Phase I data forthe known anticonvulsant drugs since the NINCDS had no need to attemptto detect anticonvulsant activity for these established anticonvulsantagents. Phase II-ED₅₀ data for some of the compounds are included inTable X. Table X also contains Phase II time of peak effect data for theparent anticonvulsant drugs.

The copper complex of dilantin was found to have a rapid onset andprolonged duration in inhibiting only Maximal Electroshock-inducedseizures. Dilantin is also known to only inhibit MaximalElectroshock-induced seizures but it potentiates Metrazol-inducedseizures. The copper complex did not potentiate but did block both typesof seizures. Phase II data for Cu(II)(dilantin)₂ indicate that it has atime of peak effect of 4 hours, which is longer than the time of peakeffect for dilantin (1 hour).

In Phase I studies, Cu(II)₂ (valproate)₄ appeared to be ineffectiveagainst Maximal Electroshock seizures, but had some inhibitory activityagainst Metrazol-induced seizures. This compound had a rapid onset andshort duration of activity following intraperitoneal administration and,consistently, a prolonged onset of activity at a higher dose followingsubcutaneous administration. The parent compound (valproic acid) wasalso weakly effective against Maximal Electroshock-induced seizures andmore effective against Metrazol-induced seizures.

With few exceptions the phenobarbital complexes were also found to haverapid onset and prolonged durations of activity in both models ofseizure. Although the data do not allow a rigorous comparison of thesecompounds, it is of interest that the pyridine and imidazole complexeswere somewhat less effective than the aquo complexes. All three solvateshad prolonged onsets of action following subcutaneous administration.The aquo phenobarbital complexes were most effective regardless of theroute of administration and recent data show that the tri- andpenta-aquo complexes are effective in preventing the Metrazol-inducedseizure at a dose much lower than the lowest dose routinely used as thelowest dose in Phase I studies, 30 mg/kg. Activity at 5 mg/kg wouldappear to indicate greater activity than phenobarbital which has an ED₅₀of 13 mg/kg.

Copper(II)(amobarbital)₂ complexes also appear to have rapid onsets andshort durations of activity following intraperitoneal administration,which appears to be reversed with subcutaneous administration.

The copper complex of lorazepam appears to have a rapid onset of actionand prolonged duration following intraperitoneal and subcutaneousadministration. This complex appears to be quite active. However, thiscomplex involves complexation at the 4-nitrogen and its stability maynot be as high as others. The copper complex of diazepam also appears tohave a rapid onset of action and prolonged duration followingintraperitoneal administration.

Cu(II)α-methyl-α-phenylsuccinimide and Cu(II)5-ethyl-5-phenylhydantoincomplexes were also effective in preventing both types of seizure withrapid onsets of action and prolonged durations of action followingsubcutaneous and intraperitoneal administration.

Cu(II)oxazepam also protected against both types of seizure and seems tobe more prolonged in its action against Metrazol induced seizures.

The Cu(II)carbamazepine complex was also an effective anticonvulsant.However, it did not potentiate Metrazol seizures while the parent drugis known to be a seizure-inducing agent.

Cu(II)(clonazepam)₂ was found to be a very potent anticonvulsant. Dosesless than those used in routine Phase I studies were required to obtainthe lowest effective doses in both models of seizure. In addition, theresults of Phase II studies with this complex show that the complex isfour times as effective as the parent drug.

The copper complexes of thiopental were tested at lower doses thanusually used in Phase I studies. It is anticipated that higher doses ofthese complexes will evidence anticonvulsant activity.

Since all of the foregoing drugs (parent compounds or ligands) are knownto be active anticonvulsants, simultaneous comparisons of the ligandsand their copper complexes are ultimately required to determine whetheror not these complexes are more active than the ligands, as suggested bysome of the data. Nevertheless, the data in Table X do indicate that allthe copper complexes tested have anticonvulsant activity. Even if someof the copper complexes are not more active than the parent compound,they may nonetheless prove useful in therapy regimens alone or inconjunction with other complexes, especially if their time of peakeffect differs from that of the parent and also especially if the coppercomplexes are less toxic and associated with fewer side effects than theparent compounds.

The foregoing experiments with copper complexes of acylsalicylates,salicylates, amino acids, imines, carboxylic acids and knownanticonvulsant and antiepileptic drugs demonstrate that such complexeshave anticonvulsant activity. That the intact copper complex may play akey role in the observed anticonvulsant activity is consistent with theobservation that inorganic copper salts, which contain much more copperon a weight percentage basis, do not have anticonvulsant activity, orhave less anticonvulsant activity based upon copper content, and alsowith the observation that there is a lack of an apparent directcorrelation between the observed anticonvulsant activity of coppercomplexes and the amount of copper in them.

The organic compounds of copper or their solvates and other chemicalmodifications useful in the present invention can be administered assolid, solution, suspension, or ointment-dosing formulations in aconcentration range of between about 0.01 to about 600 mg/kg of bodyweight and preferably between about 0.01 to about 100 mg/kg of bodyweight. The compounds can be administered orally, topically orparenterally, that is, intraperitoneally, subcutaneously orintravenously.

Having described the invention with particular reference to thepreferred form thereof, it will be obvious to those skilled in the artto which the invention pertains after understanding the invention, thatvarious changes and modifications may be made therein without departingfrom the spirit and scope of the invention as defined by the claimsappended hereto.

I claim:
 1. A method of treating convulsive tremors or convulsiveseizures comprising administration to a mammal of a therapeuticallyeffective amount of an organic compound of copper having anticonvulsantactivity selected from the group consisting of a copper complex of animinostilbene, a copper complex of a benzodiazepine, a copper complex ofa sulfonamide, and a mixture thereof.
 2. A method of treating epilepsycomprising administration to a mammal of a therapeutically effectiveamount of an organic compound of copper having antiepileptic activityselected from the group consisting of a copper complex of animinostilbene, a copper complex of a benzodiazepine, a copper complex ofa sulfonamide, and a mixture thereof.
 3. The method according to claim 1or 2 wherein the organic compound of copper is selected from the groupconsisting of:(a) Cu(II)carbamazepine; (b) Cu(II)(clonazepam)₂ Cl₂ ; and(c) Cu(II)oxazepam.
 4. The method according to claim 1 or 2 wherein theorganic copper compound is administered intravenously.
 5. The methodaccording to claim 1 or 2 wherein the organic copper compound isadministered intraperitoneally.
 6. The method according to claim 1 or 2wherein the organic copper compound is administered subcutaneously. 7.The method according to claim 1 or 2 wherein the organic copper compoundis administered orally.
 8. The method according to claim 1 or 2 whereinthe organic copper compound is administered topically.
 9. The methodaccording to claim 1 or 2 wherein the organic copper compound isadministered in an amount between about 0.1 to about 100 milligrams perkilogram of body weight.
 10. The method according to claim 1 or 2wherein the organic copper compound is administered in the form of asolution, suspension or ointment.
 11. The method according to claim 10wherein the solution, suspension or ointment includes saline, ethyleneglycol or a suspending agent like polyoxyethylene glycol.